Conifers have evolved sophisticated terpenoid defenses for protection against herbivores and pathogens. Pinus massoniana Lamb. is the most widely distributed pioneer afforestation and resin tree species in China, but is seriously harmed by pine wilt disease. Monochamus alternatus is the main vector of pine wilt disease in China. Monoterpenes, sesquiterpenes and diterpenes, the main secondary defensive compounds of P. massoniana, are catalyzed by different terpene synthases (TPSs), which participate in the important defense pathways against external biotic and abiotic stresses. Here, we aimed to identify the terpene synthases (TPSs) in P. massoniana, responding to the feeding of M. alternatus, and to characterize the functions and products of the mono-TPSs. We identified six differentially expressed TPS genes in the P. massoniana fed upon by M. alternatus, including four mono-TPS and two sesqui-TPS genes. The functions of the four mono-TPSs were verified by analysis of the main product and by-products of these mono-TPSs. (+)-α-Pinene, (−)-α-pinene, and limonene were the major products of TPS (+)-α-pinene, TPS (−)-α-pinene, and TPS limonene, respectively, but TPS (−)-β-pinene only catalyzed a trace amount of (−)-β-pinene in the products. Our findings shed light on the potential relationships between the structure of terpene synthases and their corresponding products.

Background: Pinus massoniana is a significant lipid-producing tree species in China and a susceptible host for both the pine wood nematode and its insect vector, Monochamus alternatus. The basic helix–loop–helix (bHLH) family of transcription factors play a crucial role in responding to both biotic and abiotic stresses. However, the role of bHLH in terpene-induced defense in P. massoniana remains poorly studied. Results: Transcriptome sequencing using DNA Nanoball Sequencing (DNBSEQ) and PacBio Sequel platforms was performed, revealing differences in gene expression in P. massoniana branch under the simulated feeding treatment of methyl jasmonate (MeJA) spraying. Fifteen bHLH genes were cloned and analyzed, among which eight highly upregulated PmbHLH genes showed similar temporal expression after MeJA treatment and M. alternatus adult feeding. Five highly upregulated bHLH genes with nuclear localization were highly expressed in P. massoniana after M. alternatus feeding and interacted with the promoter of the terpene synthase gene Pm TPS (−)-α-pinene, confirming their involvement in the defense response of P. massoniana against the M. alternatus adult feeding. Conclusions: Our results unveil the temporal changes and the regulation of the induced defense system in P. massoniana mediated by both MeJA signaling and M. alternatus feeding treatment. The potential application for transgenic experiments and the breeding of resistant species in the future were discussed.

The Asian longhorn beetle (ALB), Anoplophora glabripennis, and citrus longhorn beetle (CLB), Anoplophora chinensis, are two destructive invasive wood-boring pests, with high similarities in morphology, geographical distribution, host range, life cycle, adult behaviors and male-produced pheromone, implying a potential existence of interspecific interactions. Matings have been found to occur across females and males of the two species when manually paired in confined spaces. However, interspecific mating and its regulating factors are unclear between sympatric populations on hosts. Herein, by observing mountings and tracking the beetles that freely coexisted on host branches in cages, we found that the majority of mountings appeared within species; however, interspecific mountings occasionally occurred between male CLBs and female ALBs. The CLB was more active than the ALB at night. It seems that males actively searched for female ALBs, while the inverse was the case with CLBs. The main release periods of shared pheromone components overlapped between the two species, while compound ratios had significant differences. Our results unveil a trivial and asymmetrical interspecific mating between ALBs and CLBs, implying a risk of co-outbreaks of the two species in either native or invaded areas.